ABSTRACT Each year, more than one million patients are hospitalized in the U.S. for significant skin loss due to thermal and pressure injuries, chronic diabetic ulcers or genetic skin diseases. The ability to generate engineered human skin equivalents (HSEs) has provided a promising and effective therapy for these patients. However, currently available HSEs still have various limitations including poor viability and lack of appendages or mismatch in hair density. Skin is a complex organ, which contains more than 50 different cell types and various components such as vasculature, hair follicles, sensory neurons, and immune cells. The field of skin tissue engineering and regenerative medicine is currently limited by the lack of skin models that recapitulate the anatomy and physiology of human skin. This research will advance the generation of high-fidelity engineered skin grafts with functional appendages to be used in skin replacement therapy as well as modeling skin disorders in 3D. Our recent work has improved the viability of skin grafts by establishing a method to micropattern vasculature in HSEs, however, it still remains a prevailing challenge to incorporate hair follicles into engineered skin grafts in order to improve wound healing and skin function. The goal of this project is to engineer viable and functional skin equivalents with hair follicles and vasculature, using exclusively cultured human cells, for future use in drug screening and clinical applications. These studies will address several prevailing challenges in the field of skin/hair tissue engineering by achieving: i) efficient hair induction using cultured human DPC; ii), initiation of hair morphogenesis in 3D human skin equivalents in vitro, iii) control over region-specific hair density, iv) viable skin grafts through promoting adequate vascularization, and v) combination of a vasculature network with hair follicles to build increase the complexity of existing 3D skin models. This project is highly responsive to several of the areas of interest in this RFA-AR-17-005 including: 1) Sophisticated skin models that incorporate multiple skin components such as hair follicles, vasculature, nerves, sweat glands, immune cells, and fat; 2) Add complexity to existing model systems; and 3) Engineering functional stem cell niches in 3-D musculoskeletal and skin models. We will employ bioengineering, genetics, pharmacological and systems biology approaches to address this hypothesis, allowing for the development of truly functional and viable skin substitutes for patients with significant skin/hair loss, and providing 3D in vitro screening models of skin and hair follicles for pharmaceutical development applications.